MyD88 is an adaptor protein involved in interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) induced activation of NF-κB (Burns et al. 1998, J. Biol. Chem. 273:12203-12209; Wesche et al. 1997, Immunity 7:837-847; Medzhitov et al. 1998, Mol. Cell. 2:253-258). Both human and mouse MyD88 proteins are composed of a C-terminal Toll/IL-1R homology (TIR) domain and an N-terminal death domain (DD). These domains are separated by a small intermediate domain (ID). MyD88 links the TIR domain of IL-1R/TLR with the DD of the Ser/Thr kinase, IRAK-1. MyD88 also recruits IRAK-4 via its ID. This enables IRAK-4 to phosphorylate IRAK-1. Phosphorylated IRAK-1 then interacts with TRAF6, which in turn, transmits a signal leading to activation of the IκB kinase complex and JNK (Cao et al. 1996, Nature 383:443-446). This eventually results in the activation of transcription factor NF-κB. As MyD88 is known to play a central role in regulating signaling through IL-1 and Toll-receptors, various strategies have been used to demonstrate that inhibition of MyD88 function results in an alleviation of inflammatory responses. For example, in vitro this has been clearly demonstrated using MyD88 deficient cells (Schnare et al. 2000, Curr. Biol. 10: 1139-1142; Yamamoto et al. 2002, Nature. 420:324-329). In animals, genetic ablation of MyD88 produces a diminution of responses to IL-1β and an overall reduction in models of inflammation (Kawai et al. 1999, Immunity 11:115-122; Bjorkbacka et al. 2004, Nature Med. 10:416-421; Adachi et al. 1998, Immunity 9:143-150). MyD88 deficient mice demonstrate reduced pathology associated with polymicrobial sepsis (Weighardt et al. 2002, J Immunol. 169:2823-2827) and defects in T-cell proliferation and induction of acute phase proteins in response to IL-1β (Adachi et al. 1998, Immunity 9:143-150).
A lipopolysaccharide (LPS)-induced splice variant of MyD88, MyD88S, which has been reported to function as a dominant-negative regulator of IL-1β- and LPS-induced NF-κB activation, has recently been identified (Janssens et al. 2002, Curr. Biol. 12:467-471). The MyD88 gene contains 5 exons (Hardiman et al. 1997, Genomics. 45:332-339). MyD88S is produced as the result of complete excision of exon II from the mature mRNA by alternative splicing. This leads to an in-frame deletion of the complete ID (amino acids 110-154). Although MyD88S still binds IL-1R and IRAK-1, it is defective in its ability to recruit IRAK-4 and induce subsequent IRAK-1 phosphorylation and NF-κB activation (Burns et al. 2003, J. Exp. Med. 197:263-268). The ability of MyD88S to function as a dominant negative regulator of IL-1β and LPS signaling suggests that strategies to modify MyD88 RNA splicing, such that MyD88S is expressed preferentially to the larger MyD88 isoform (referred to herein as MyD88L), would be therapeutically valuable in treating inflammatory diseases associated with excessive IL-1R signaling.
Although variations in alternative splicing are widely recognized as a mechanism to generate molecular diversity and clearly contribute to certain diseases (Maniatis and Tasic, 2002, Nature. 418:236-243; Johnson et al. 2003, Science. 302:2141-2144; Yeo et al. 2004, Proc. Natl. Acad. Sci. U.S.A. 101:15700-15705), the development of pharmacological agents capable of controlling mRNA splicing has remained challenging. An approach that has recently gained significant acceptance is the use of chemically modified antisense oligonucleotides. When appropriately designed and directed to hybridize to RNA sequences adjacent to splice junctions, these have been shown to effectively modulate pre-mRNA splicing (Sazani and Kole, 2003, J. Clin. Invest. 112:481-486; Taylor et al. 1999, Nat. Biotech. 17:1097-1100; Garcia-Blanco et al. 2004, Nat. Biotech. 22:535-546).
U.S. Pre-Grant Publication 2005-0181476 discusses the identification of MyD88S and methods of modulating alternative splicing of MyD88 through RNA inhibition and use of antisense oligonucleotides.
A method of controlling the behavior of a cell through modulation of the processing of an mRNA target by contacting the cell with an antisense compound acting via a non-cleavage event is disclosed in U.S. Pat. No. 6,210,892 and U.S. Pre-Grant Publication 2002-0049173.
Taylor et al. (1999, Nat. Biotech. 17:1097-1100) and Karras et al. (2000, Mol. Pharm. 58:380-387) describe modified oligonucleotides for modulation of pre-mRNA splicing of Bcl-x and interleukin-5 receptor alpha, respectively.
Kole et al. (WO 94/26887 and U.S. Pat. Nos. 5,627,274; 5,916,808; 5,976,879; and 5,665,593) disclose methods of combating aberrant splicing using modified antisense oligonucleotides which do not activate RNase H.
Antisense technology is an effective means for modulating expression of one or more specific gene products and is uniquely useful in a number of therapeutic, diagnostic, and research applications. Provided herein are modified antisense compounds for use in modulation of MyD88 splicing.